Analysis of blood samples often requires separation of whole blood into a serum or plasma fraction and a cell-containing fraction prior to assay. Typically this is performed by collecting a blood sample in a blood collection or separation tube. Ideally such a blood collection or separation tube should be sterilizable in order to avoid microbial contamination of the sample and to simplify long term storage. Since blood collection/separation tubes are produced in large numbers and are provided as sealed containers with reduced internal pressure (in order to facilitate blood collection) such sterilization is typically carried out using ionizing radiation, such as gamma or e-beam irradiation, or other methods that permit sterilization of the contents of closed containers. Following sample collection, the blood collection or separation tube is transferred to a centrifuge where it is spun at relatively high speed. This application of centripetal force generates a density gradient within the tube, with heavier elements of the blood sample (for example, blood cells) collecting in the bottom of the tube as a dense phase or fraction while lighter elements (for example, serum or plasma) collect towards the top as a light phase or fraction. Following removal from the centrifuge the separation tube may be moved to an analyzer or, alternatively, placed in storage.
Unfortunately, once blood is separated in this manner these phases or fractions can remix through diffusion, agitation, disturbance during sample extraction or removal from storage, or other undesirable interactions. The situation is exacerbated by rupture of cells within the dense cell-containing fraction on storage. This can lead to contamination between the fractions, which can adversely impact assay performance. Ideally, therefore, blood fractions should remain isolated from one another following separation to ensure that no contamination occurs prior to or during analysis.
Systems that isolate the whole blood fractions generally include a separator substance or device that has a density intermediate between that of the cell-containing fraction and the serum/plasma fraction of whole blood, which allows the separator substance or device to localize between these fractions spontaneously during centrifugation. This allows the separator substance or device to act as a physical barrier to mixing due diffusion, agitation, and other disturbances. A suitable density is typically between 1.01 g/cm3 and 1.09 g/cm3. When whole blood is added to a collection or separator tube containing a separator substance and the tube is centrifuged, the separator substance migrates to the interface between the fractions thereby isolating the two fractions from each other. An exemplary collection tube that utilizes a flowable gel as a separator substance in the fractionation of whole blood can be found in U.S. Pat. No. 4,946,601 (to Fiehler). Another example of a separator substance that is flowable in the preparation of fractions from whole blood can be found in U.S. Pat. No. 6,248,844 and U.S. Pat. No. 6,361,700 (to Gates et al). In these patents the separator substance is a polyester that is cured to achieve a desired viscosity. These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
Although providing a flowable substance allows for separating the fractions of whole blood, flowable substances have several disadvantages. A flowable substance still remains flowable after centrifugation is complete. A result of this physical instability is that there remains a risk of contamination between the fractions of the sample unless proper care is taken to keep the blood collection tube suitably still and protected from agitation. While it is possible to use a gel-based separation substance that is formulated or configured with a viscosity that is high enough to provide a sufficiently solid barrier to overcome these disadvantages, such a separation substance may no longer be suitably flowable with whole blood and therefore require prohibitively long centrifuge times. Short centrifuge times are important for maintaining high throughput in the clinical laboratory and may be critical in life or death situations where a blood analysis result is required quickly.
One approach to resolving this issue is to utilize a thixotropic gel as a separator substance in a blood collection tube. Thixotropic gels have viscosities that change depending upon the shear stress applied to them. For example, a thixotropic gel may have a low viscosity that allows it to flow relatively easily when under the shear force applied by a centrifuge, but may have a high viscosity that makes it resistant to flow when it is at rest following centrifugation. U.S. Pat. No. 4,818,418 (to Saunders), for example, discusses the use of thixotropic gels in blood collection tubes. The problem with thixotropic gels, however, is that the relatively viscous gel that localizes between the blood fractions remains at least somewhat flowable and does not form a sufficiently solid separation barrier. As a result, when a sample is extracted from the tube with a pipette or similar device it is possible for such a separation substance to contaminate, foul, or plug the pipette if it contacts such a separation barrier. Moreover, it is also not uncommon that pieces of the separation substance float in the upper phase and cumulatively plug probes of an analyzer. While most analyzers have depth sensors, such sensors do not solve the problem of floating separator substances, and also waste plasma as use of such sensors require clearance above the separator substances.
An alternative approach taken by some medical device manufactures is to place a moveable, solid barrier substance or device within the blood collection tube. Examples of such solid substances include the intermediate density polymers found in U.S. Pat. No. 3,647,070 (to Adler) where polymer spheres or granules of a specified density aggregate to form a barrier layer. Similarly, U.S. Pat. No. 5,266,199 (to Tsukagoshi et al) describes an elastic tube and ball valve assembly that controls separation of the serum from the cell-containing phase. Such physical barriers, however, necessarily include gaps between the individual components and therefore only provide a partial seal between the separated blood fractions. Moreover, a further disadvantage to a movable barrier is that such containers must be filled adequately and allow for differences in relative proportions of fractions.
Yet another approach to providing a solid barrier between fractions in a blood collection/separator tube is disclosed in U.S. Pat. No. 7,673,758, U.S. Pat. No. 7,674,388, U.S. Pat. No. 7,780,861, U.S. Pat. No. 8,151,996, and U.S. Pat. No. 8,206,638 (to Emerson). These disclose the use of a thixotropic gel within a blood collection or separator tube that has a low viscosity during centrifugation, allowing it to flow relatively freely and localize between the dense cell-containing fraction and the relatively low density serum/plasma fraction during centrifugation. The thixotropic gel includes a polymer with reactive groups that are capable of forming chemical crosslinks and a polymerization initiator. Activation of the initiator following centrifugation causes the reactive groups of the polymer to form covalent bonds that crosslink polymer molecules within the gel. This crosslinking forms a solid, impermeable polymer barrier between the cell-containing and serum/plasma blood fractions. The problem of adequately sterilizing a blood collection or separator tube containing such a composition, however, is not addressed.
These and other solutions for whole blood separation lack the necessary features to ensure that both the separated fractions of whole blood are effectively protected against contamination due to microbial growth and undesirable interactions between sample fractions/phases while supporting short centrifugation times. Thus, there is still a need for liquid separation technologies in which the separation barrier is solidified after centrifugation and that allow for effective and economical sterilization of devices incorporating them.